{"id":610931,"date":"2024-06-10T20:00:00","date_gmt":"2024-06-11T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/red2flpe-scon-a-versatile-multicolor-strategy-for-generating-mosaic-conditional-knockout-mice-nature-communications\/"},"modified":"2024-06-11T04:25:41","modified_gmt":"2024-06-11T08:25:41","slug":"red2flpe-scon-a-versatile-multicolor-strategy-for-generating-mosaic-conditional-knockout-mice-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/red2flpe-scon-a-versatile-multicolor-strategy-for-generating-mosaic-conditional-knockout-mice-nature-communications\/","title":{"rendered":"Red2Flpe-SCON: a versatile, multicolor strategy for generating mosaic conditional knockout mice – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
We previously reported the Red2Onco system<\/i> \u2014 a series of modified Rosa26-Confetti (Brainbow2.1) alleles, which enable the mosaic, ectopic expression of oncogenes in a red fluorescent protein (RFP)-labeled, clone-specific manner22<\/a><\/sup>. We adapted an efficient electroporation protocol to achieve the desired gene knock-in, adjacent to the RFP, using CRISPR-Cas9 nickases with Confetti embryonic stem cells (ESCs); a process that usually takes approximately two to three weeks (Supplementary Fig. 1<\/a>). The targeting approach harnesses the homology-directed repair (HDR) pathway, with homology arms of 600\u2013700\u2009bp in length. The Red2 targeting vector contains specific restriction cloning sites for inserting the cDNA sequence \u2014 downstream and in frame with the RFP and 2A peptide \u2014 and a PGK-Blasticidin-pA cassette with an inverted orientation for efficient antibiotic selection (Supplementary Fig. 1a<\/a>). The RFP protein, tdimer2<\/i>, of the confetti allele consists of two segments with repeated sequences. The left homology arm contains the sequence of one dimeric unit and can therefore be inserted to replace either dimer (with about 650\u2009bp difference in length), where the second dimer is the desirable target24<\/a><\/sup> (Supplementary Fig. 1b<\/a>). For the targeting experiment, we utilized a pair of sgRNAs with a Cas9-D10A nickase (which only cleaves strands that are complementary to the sgRNA) to minimize any potential off-target effects25<\/a><\/sup> (Supplementary Fig. 1c<\/a>). Upon electroporation, a small fraction of cells showed GFP expression from the Cas9 nickase vectors (Supplementary Fig. 1d<\/a>). After 48\u201372\u2009h of recovery, cells were subjected to blasticidin treatment to select for targeted clones (Supplementary Fig. 1e<\/a>), which were checked by long range PCR genotyping. Verified clones were then injected into developing blastocysts to generate chimeras (Supplementary Fig. 1f<\/a>). This CRISPR-mediated method of targeting enabled mosaic genetic Red2cDNA mouse lines to be generated efficiently.<\/p>\n Here, we present a mosaic knockout system that involves the insertion of the Flpe recombinase sequence26<\/a><\/sup> adjacent to an RFP (tdimer2) linked with a P2A peptide. This setup allows for the RFP-labeled (red), clone-specific, Flpe-mediated recombination of the target FRT allele, while ensuring that all the other fluorescent proteins remain genetically unaltered (Fig. 1a, b<\/a>). We named this system, Red2Flpe<\/i>. The mosaic knockout system, induced upon Cre-mediated recombination, results in the presence of both wild-type and mutant knockout cells in the same tissue. The YFP-labeled wild-type cells and RFP-labeled mutant cells undergo clonal competition that involves the secretion of paracrine factors (Fig. 1b<\/a>). Depending on how the target gene knockout impacts the clonal fitness, the mutant cells could either outcompete or become eliminated, correspondingly (Fig. 1b, c<\/a>). Cells carrying cancer driver mutations are known to secret paracrine factors to suppress the growth of neighboring cells22<\/a>,27<\/a>,28<\/a><\/sup>, Red2Flpe is therefore superior in accurately tracking the evolution of mutant cells and the wild-type cells in the same tissue with spatiotemporal resolution (Fig. 1c<\/a>).<\/p>\n a<\/b> Upon Cre induction, each cell (unless it is polyploid) is adapted to express one fluorescent protein color. The cells labeled with red fluorescent protein (RFP) express the Flpe recombinase, while all the other fluorescent protein colors (green\/yellow\/cyan) correspond to wild-type cells. b<\/b> Schematic showing the interactions between YFP+ wild-type cells (GeneX<\/i>WT\/WT<\/i><\/sup>) and RFP+ mutant cells (GeneX<\/i>\u2206\/\u2206<\/sup>) through paracrine signaling. As a result, the clonal competition between wild-type and mutant cells can be quantified. c<\/b> Wholemount intestines of Tg(Vil-CreERT2<\/sup>); Red2Flpe; ApcFRT\/FRT<\/sup> mice from 2 and 15 weeks after tamoxifen administration. The mutant RFP cells, through the secretion of paracrine factors, outcompete the surrounding wild-type cells. The experiment in c<\/b> was performed in 3 separate Tg(Vil-CreERT2<\/sup>); Red2Flpe; ApcFRT\/FRT<\/sup> mice for each time point and the representative images were taken. Scale bar, 50\u2009\u03bcm.<\/p>\n<\/div>\n<\/div>\n After activating Red2Flpe<\/i> with the ubiquitous Rosa-CreER<\/i>T2<\/i><\/sup> line, we harvested the colon, pancreas, seminal vesicles, spleen, stomach and tongue (Supplementary Fig. 2a\u2013f<\/a>), and confirmed that both YFP and RFP were expressed in all of the analyzed tissues. Quantification of YFP+ and RFP+ clones showed no recombination bias for one fluorescent marker versus the other, as similar numbers of both clones were scored (Supplementary Fig. 2g, h<\/a>). This demonstrated the utility of Red2Flpe<\/i> in multiple organs and tissues with recombination rates that were suitable for mosaic lineage tracing studies.<\/p>\n We next tested whether the expression of Flpe on the modified Confetti allele could recombine with the FRT sites on different alleles in the mouse genome, in a red clone-specific manner. In combination with the intestinal epithelium-specific CreERT2<\/sup> line, Villin-CreERT<\/i>2<\/a><\/sup>, we crossed Red2Flpe<\/i> with two separate FRT lines. Firstly, Vil-CreER<\/i>T2<\/i><\/sup>;Red2Flpe<\/i> was crossed with a newly generated Apc-FRT (exon 15 flanked by FRT sites) (Fig. 2a<\/a>); knockout of Apc leads to stabilized \u03b2-catenin accumulation and subsequent Wnt signaling activation29<\/a><\/sup>, similar to the inactivation of other Wnt negative regulators30<\/a>,31<\/a><\/sup>. One week after the Cre-mediated labeling (Fig. 2b<\/a>), the Vil-CreER<\/i>T2<\/sup>;Red2Flp<\/i>;Apc<\/i>FRT\/FRT<\/i><\/sup> mouse intestine showed that the RFP clones, specifically, displayed elevated cytoplasmic \u03b2-catenin staining (Fig. 2c, d<\/a>). Secondly, the Vil-CreER<\/i>T2<\/i><\/sup>;Red2Flpe<\/i> line was crossed with an FRT-based GFP reporter line called RCE:FRT<\/i> (an FRT-stop-FRT GFP reporter on the Rosa26 locus)32<\/a><\/sup> (Fig. 2e<\/a>). We expected overlapping GFP and RFP signals but no overlap with any other colors, if Flpe worked efficiently and specifically. After one week of Cre induction, we only observed GFP expression in the Flpe-expressing RFP+ cells (Fig. 2f\u2013h<\/a>). The rate of accumulation of RFP\/GFP double positive clones was comparable to other established Confetti-based reporters8<\/a><\/sup>, showing a minimal delay between RFP expression and Flpe-dependent GFP expression. To assess the efficiency and specificity of Flpe\/FRT recombination of Red2Flpe, primary intestinal cells were harvested and analyzed by flow cytometry at five, seven and ten days after tamoxifen injection (Fig. 2i\u2013k<\/a>). A stringent gate was set for the RFP-positive cells, as the maturation time is longer for tdimer2 (~120\u2009min)24<\/a><\/sup> (Fig. 2i<\/a>). We observed an increase in the proportion of GFP\/RFP double-positive cells from day five (66.3%) to day seven (82.1%) and day ten (82.6%) (Fig. 2j<\/a>). There is a time lag between the point of tamoxifen injection, the observed Cre activity and the subsequent stable expression of the fluorescent reporters and Flpe recombinase; this data, therefore, suggests that Red2Flpe becomes active approximately five to seven days after the tamoxifen injection (Fig. 2j<\/a>). Overall, these results indicate that Red2Flpe<\/i> is a functional and efficient genetic tool that can be used to generate mosaic multicolor mouse models in vivo.<\/p>\n a<\/b>\u2013d<\/b> Small intestine samples harvested one week after treatment with tamoxifen (2\u2009mg tamoxifen per 20\u2009g body weight) from a Tg(Vil-CreERT2<\/sup>); Red2Flpe; ApcFRT\/FRT<\/sup> mouse. The wild-type crypts labeled with either cyan (CFP), yellow (YFP), or no color show clear \u03b2-catenin staining in the membrane with less staining in the cytoplasm. The RFP-labeled (red) crypts display an increase in the cytoplasmic fraction of \u03b2-catenin staining \u2014 indicating successful knockout of Apc<\/i> and malfunction of the \u03b2-catenin destruction complex. The RFP-labeled (red) crypts and the corresponding \u03b2-catenin staining are marked with dotted lines. The experiment in a<\/b>\u2013d<\/b> was performed in 3 separate Tg(Vil-CreERT2<\/sup>); Red2Flpe; ApcFRT\/FRT<\/sup> mice the representative images were taken after antibody staining. Scale bar, 50 \u03bcm. e<\/b>\u2013h<\/b>, Small intestine samples harvested one week after treatment with tamoxifen (2\u2009mg per 20\u2009g body weight) from a Tg(Vil-CreERT2<\/sup>); Red2Flpe; Gt(ROSA)26Sortm1.2(CAG-EGFP)Fsh<\/sup> mouse. The expression of RFP (red) coincides with GFP (green) but does not coincide with Confetti YFP (yellow). RFP-GFP double positive cells are marked by the triangles and the YFP cells are marked by the arrows. The experiment in h<\/b> was performed in 2 separate Tg(Vil-CreERT2<\/sup>); Red2Flpe; Gt(ROSA)26Sortm1.2(CAG-EGFP)Fsh<\/sup> mice the representative images were taken. i<\/b> Gating strategy of YFP, RFP and CFP cells in tamoxifen-induced Tg(Vil-CreERT2<\/sup>); Red2Flpe intestine. j<\/b> GFP+ cells within the gated CFP+ population (top row) or the gated RFP+ population (bottom row) in an FRT-based reporter (Tg(Vil-CreERT2<\/sup>); Red2Flpe; Gt(ROSA)26Sortm1.2(CAG-EGFP)Fsh<\/sup>) mouse harvested 5, 7 or 10 days post tamoxifen administration. k<\/b> GFP+ cells in the control mouse (Tg(Vil-CreERT2<\/sup>); Red2Flpe) after tamoxifen administration. For each time point in i<\/b> and j<\/b>, the intestines from 2 mice of Tg(Vil-CreERT2<\/sup>); Red2Flpe; Gt(ROSA)26Sortm1.2(CAG-EGFP)Fsh<\/sup> and one control mouse of Tg(Vil-CreERT2<\/sup>); Red2Flpe were profiled and showed similar results.<\/p>\n<\/div>\n<\/div>\n As Cre-loxP was found to work more efficiently than the wild-type Flp, most of the murine conditional knockout (cKO) lines were made using the Cre-loxP system. Conditional mouse lines are often generated by in vitro ESC targeting, followed by a blastocyst injection to acquire chimeric mice, and final germline transmission; this process takes at least six months. Despite efforts to improve the efficiency of the zygote injections, using CRISPR-based knock-in with a long single-stranded oligonucleotide (ssODN)33<\/a><\/sup>, the process remains technically challenging.<\/p>\n To facilitate cKO mouse generation, we recently developed an artificial intron-based approach that uses a S<\/u>hort C<\/u>onditional intrON (SCON), which is just 189\u2009bp in length23<\/a><\/sup>. This method only requires a synthesized oligo template, a synthesized gRNA, and a commercially available Cas protein and\/or mRNA. All these components are injected into zygotes to generate a cKO. SCON has a neutral effect following the initial insertion of the target gene, and induces the expected loss of function effect upon recombination in vivo. SCON, therefore, offers an alternative but efficient way to generate cKO alleles using a method that is as simple as CRISPR-based \u2018tagging\u2019.<\/p>\n We reasoned that the loxP recombination sites used with SCON could be exchanged with FRTs, for compatibility with the Red2Flpe mosaic knockout system. The SCON-FRT system works in the same way as the SCON-loxP system \u2014 which consists of a splice donor, branch point, and splice acceptor \u2014 with SCON acting as a functional intron. With this system, two FRT recombination sites flank the branch point; the removal of the branch point upon Flp-mediated recombination then abrogates the SCON\u2019s intronic function, causing it to be retained in the mature transcript after splicing. The remaining 55\u2009bp SCON intron sequence contains potential stop codons that can cause premature termination of translation and subsequent truncation of the target protein (Fig. 3a<\/a>).<\/p>\n a<\/b> Schematic diagram of SCON-FRT in an eGFP overexpression construct, including eGFP, eG-SCON-FRT-FP and recombined eG-SC-FRT-FP. SD (splice donor), BP (branch point), SA (splice acceptor). b<\/b> Brightfield and fluorescent images of HEK293T cells 24\u2009h after transfection. c<\/b> GFP fluorescence level of HEK293T cells 48\u2009h after transfection. Red: intact eGFP; Blue: eG-SCON-FRT-FP; Orange: recombined eG-SC-FRT-FP. d<\/b> GFP fluorescence level of HEK293T cells with integrated eG-SCON-FRT-FP before (blue) and after (orange) transfection of a Flp-expressing plasmid. Experiments in c<\/b> and d<\/b> were performed twice and in two separate cell clones, which showed similar results. Violin and box plots indicate the distribution of data where minimum and maximum values are presented. The boxplot represents the central 50% of data points and the thickened line marks the median value. Source Data relevant to this Figure are provided with this paper in Source Data file.<\/p>\n<\/div>\n<\/div>\n We next tested whether the SCON-FRT system had a similar neutral effect on gene expression, as with SCON-loxP. We transfected HEK293T cells with either intact eGFP; eG-SCON-FRT-FP (eGFP with a SCON-FRT insertion); or the recombined form, eG-SC-FRT-FP (Fig. 3b<\/a>). We found that the intact eGFP and the two different eG-SCON-FRT-FPs, with either wild-type or F3 FRT sites, had comparable GFP levels; however, the wild-type FRT version slightly out-performed the F3 FRT version (Fig. 3c<\/a>). With the recombined forms, the level of GFP fluorescence was not detectable, indicating loss of expression (Fig. 3b, c<\/a>). We also tested whether SCON-FRT could be efficiently recombined in mammalian cells upon Flp expression. We generated mouse ESCs with constitutive expression of eG-SCON-FRT-FP, using the piggyBac transposon system. After transfecting an Flp-expressing plasmid, we found that the level of GFP diminished significantly, which confirmed the compatibility of the SCON-FRT for Flp\/FRT-based recombination system in mammalian cells (Fig. 3d<\/a>). Therefore, we concluded that the SCON-FRT system was also neutral, like the SCON-loxP system, and it was applicable in mammalian cells.<\/p>\n Next, we assessed the compatibility of the SCON-FRT system with Red2Flpe<\/i>. We made use of Confetti<\/i> and Red2Flpe<\/i> ESCs in combination with piggyBac-eG-SCON-FRT-FPs (Fig. 4<\/a>a, b). Cells with an integrated eG-SCON-FRT-FP were selected with puromycin with the expectation that both eGFP and puromycin-resistance expression would be compromised following Flp-mediated FRT recombination (Fig. 4a, b<\/a>). After selecting GFP-expressing cells, we induced the recombination of the Confetti<\/i> and Red2Flpe<\/i> alleles, respectively, by transfecting a Cre-expressing plasmid. Using fluorescent activated cell sorting (FACS), RFP+ cells were sorted and cultured separately from the uninduced cells. All the RFP+ colonies of Red2Flpe<\/i> ESCs showed no eGFP expression compared to the Confetti<\/i> ESCs, which were used as controls (Fig. 4c<\/a>). Flow cytometry revealed that most of the cells in the uninduced cultures retained high levels of GFP expression, despite some transgene silencing, while all of the recombined RFP+ cells lost GFP expression completely (Fig. 4d<\/a>). These results indicated that Red2Flpe could be coupled with SCON-FRT to successfully achieve conditional mosaic gene knockouts.<\/p>\n a<\/b> Schematic of the experimental set up that involves in integration of a piggybac-eG-SCON-FP vector into the Confetti<\/i> or Red2Flpe<\/i> embryonic stem cells. Upon Cre recombination, the RFP\u2009+\u2009GFP+ cells are sorted and cultured in puromycin-containing media. To assess the recombination, and the loss of GFP signals, puromycin was omitted from the media. b<\/b> Schematic of the piggybac vector carrying the overexpression cassette of eG-SCON-FRT-FP coupled with puromycin resistance. Both eGFP and puromycin expression are reduced following Flp\/FRT recombination. c<\/b> Brightfield and fluorescent images of RFP+ Confetti and Red2Flpe ESCs with integrated eG-SCON-FRT-FP. The uninduced clone that retains eGFP expression is marked by an arrow. d<\/b> GFP fluorescence level of Red2Flpe ESCs with integrated eG-SCON-FRT-FP after puromycin withdrawal. Gray: uninduced Red2Flpe; eG-SCON-FRT-FP cells; Red: RFP+ cells. Experiment shown in c<\/b> and d<\/b> was performed twice in two separate cell clones, which showed similar results.<\/p>\n<\/div>\n<\/div>\n With the knowledge that the use of SCONs would not affect basal gene expression, we generated thirteen cKO mouse lines23<\/a><\/sup>. We injected CRISPR-Cas9 ribonucleoprotein (RNP), Cas9 mRNA, and a 300\u2009bp long ssODN of SCON (using either loxP sites or FRT sites, both of which were 189\u2009bp long). We used left and right homology arms that were 55 and 56\u2009bp long, respectively23<\/a><\/sup>. With the SCON approach, a complete experimental mouse line for mosaic knockout studies could be generated quickly and easily.<\/p>\n We propose a pipeline for generating zygotes using SCON targeting from the desired CreER<\/i> and Red2Flpe<\/i> lines, both in homozygosity (Supplementary Fig. 3a<\/a>). From the offspring, pups that are heterozygous for the SCON-FRT knock-in can be used for mating to create further experimental lines (Supplementary Fig. 3b<\/a>). The chance of acquiring the desired experimental cohort is 1\/4 (Supplementary Fig. 3c<\/a>) so, theoretically, it is possible to generate SCON-based Red2Flpe<\/i> mosaic knockout mice by zygote injection with just two mouse generations.<\/p>\n One of the first SCON-FRT lines we generated was the Sox2-SCON-FRT<\/i> (Sox2<\/i>scon<\/i><\/sup>) line (Supplementary Fig. 4a<\/a>). From twenty founder pups, we obtained three heterozygous SCON-FRT knock-in mice with precise integration (15% efficiency). Sox2<\/i>+\/scon<\/i><\/sup> mice can be bred to be homozygous without any noticeable developmental defects (refer to Fig. 4g, h in Wu et al.)23<\/a><\/sup>, which validates the utility of SCON-loxP and FRT for in vivo experiments. The mouse ESCs derived from homozygous Sox2<\/i>scon\/scon<\/i><\/sup> blastocysts expanded stably in culture. Then, following transient Flpe expression, the colonies that contained either mosaic or complete Sox2-KO cells exhibited distorted morphologies. These results were consistent with the importance of Sox2 in maintaining pluripotency (Supplementary Fig. 4b<\/a>)34<\/a><\/sup>.<\/p>\n Sox2<\/i> is a transcription factor that plays crucial roles during embryonic development \u2014 from the blastocyst stages to the fate-specifying stages \u2014 of many tissues35<\/a><\/sup>. Knockout studies revealed that Sox2<\/i> is required for proper development of the esophagus36<\/a>,37<\/a><\/sup>. In adults, Sox2<\/i> is expressed in the esophagus and stomach, and is thought to be important for stem cell maintenance38<\/a>,39<\/a><\/sup>. Consistently, tissue-wide knockout of Sox2 or depletion of Sox2-expressing cells leads to compromised tissue maintenance and physiology38<\/a>,39<\/a><\/sup>. However, as widespread knockout of Sox2 in these tissues compromises their overall integrity, the exact function of Sox2 remains unclear. A mosaic analysis of Sox2 in the adult esophagus and other tissues is therefore necessary.<\/p>\n We used Red2Flpe<\/i> and the Sox2<\/i>scon<\/i><\/sup> alleles with the ubiquitous Rosa26-CreER<\/i>T2<\/i><\/sup> inducer line, and generated Rosa26-CreER<\/i>T2<\/i><\/sup>; Red2Flpe<\/i>; Sox2<\/i>scon\/scon<\/i><\/sup> (Red2Sox2KO<\/i>) mice to investigate the function of Sox2 in the adult mouse esophagus (Fig. 5a<\/a>). By administering tamoxifen to the Red2Sox2KO<\/i> mice, we activated fluorescent labeling and the red clone-specific knockout of Sox2 (Fig. 5b<\/a>). We then lineage traced the wild-type (yellow) and knockout (red) cells and quantified the sizes of their clones over time. If Sox2 was essential for stem cell maintenance, we would have expected that the mutant clones would be rapidly lost from the basal layer within a short period of time. By contrast, if Sox2 was not essential for stem cell maintenance, we would have expected that the mutant clones would remain present in the basal layer \u2014 and might only be lost due to their relative clonal fitness in that tissue.<\/p>\nRed2-Flpe: a versatile tool that enables precise mosaic knockout with multicolor labeling<\/h3>\n
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Short Conditional intrON (SCON) facilitates the generation of FRT-based conditional knockout mouse lines with a one-step zygote injection<\/h3>\n
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Red2Flpe can be efficiently utilized in combination with the SCON-FRT system<\/h3>\n
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Sox2-SCON-FRT mouse generation via one-step zygote injection<\/h3>\n
Mosaic knockout of Sox2 in adult tissues reveals its variable essentiality<\/h3>\n